Summary: The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout in the RAW264 macrophage cell line. We initially constructed a targeting vector in which exon 12 was disrupted by a neomycin gene having its own heterologous promoter and polyadenylation signal. PCR screening of over 3000 clones indicated that no homologous recombination event had occurred. We have now constructed a promoterless construct and have shown that transfectants can be isolated. The transfectants are observed at >100-fold lower frequency, demonstrating that a more stringent selection has been achieved. PCR screening of pools of transfectants generated from the promoterless construct indicated that two pools contained clones with homologous recombinants. Clones from one of the pools have been isolated and positive clones identified by PCR. The positive clones are now being analyzed by Southern blotting to confirm the correct insertion into the genome. We plan to use a similar approach to isolate a double knockout and to compare chemotactic and other properties of the parent, single knockout and double knockout cells. The knockout and parent cells may be used to evaluate expression systems for cell activation and/or motility genes.